The rolling of human neutrophils on the endothelium is only an initial step of their extravasation, but it is a complicated dynamic process mediated cooperatively by the adhesion molecules expressed on the surfaces of endothelial cells and neutrophils, the shear stresses due to the blood flow, and the mechanical properties of the neutrophil/endothelial cell membranes and neutrophil microvilli. Therefore, the overall goal of this proposal is to investigate the details of these mechanical properties and how they will impact on the rolling process. The specific aims are to: 1) extract "single tethers to test the hypotheses that single tether formation from endothelial cells is a local phenomenon (i.e., it does not depend upon whether the cell is suspended or attached to a substrate, nor the surface receptor type used to form the tether), single tether formation from endothelial cells is modulated by stimulation, and single tethers can be extracted simultaneously from passive neutrophils and stimulated endothelial cells, 2) extract double tethers to test the hypotheses that double tether formation from human neutrophils or endothelial cells is modulated by stimulation, and double tethers can be extracted simultaneously from passive neutrophils and stimulated endothelial cells, 3) quantify the mechanical properties of a single neutrophil microvillus to test the hypotheses that the microvillus has different static properties in bending and compression compared with extension, and its viscoelastic properties are not significant during extension or compression, and 4) develop a biomechanical model to test the hypotheses that simultaneous tether formation from neutrophils and endothelial cells, compared with tether formation from neutrophils alone, further facilitates the rolling of neutrophils on the endothelium, and stimulating both neutrophils and endothelial cells, compared with stimulating neutrophils alone, also further facilitates the rolling of neutrophils on the endothelium. Tether formation from neutrophils and suspended endothelial cells will be performed with the micropipette aspiration technique, while tether formation from attached endothelial cells will be performed with the atomic force microscope. The microvillus mechanics will be studied with the optical tweezers, combined with the micropipette manipulation system.